The objective of this study is to establish a multiplexed intravital cytometry technique by combining nanocrystal-immunoconstructs and fluorescence multiphoton laser-scanning microscopy to study immune cells in tumors. In studies of the cellular immune response against tumors, the detection of cell-cell interactions, cell phenotype and intra-tumor cell distribution in orthotopic tumor models are critical to dissect the mechanisms of successful or failed immunotherapies. Unfortunately, observation of the temporal and spatial interactions of multiple cell types with traditional fluorophores is limited by their broad spectral characteristics. We propose to solve this problem by developing and optimizing infusion protocols in mice using nanocrystal-antibody complexes, which should allow us to distinguish simultaneously five or more specific probes by multiphoton laser-scanning microscopy (Aim 1). The goal is to establish and utilize targeted nanocrystal-immunoconstructs for in vivo cytometric and functional analyses of immune cells in tumors during immunotherapy (Aims 2). Establishing this multiplexed in vivo imaging technique with nanocrystal-immunoconstructs will create unique opportunities for further understanding the molecular, cellular and physiological events associated with immune responses in the stroma of living tumors or other diseased tissues, potentially translating into the discovery and validation of new therapeutic targets. In addition, the experimental system employed here has the potential to enhance cancer research in a disease - liver cancer - with dismal prognosis.) PUBLIC HEALTH RELEVANCE: Currently available intravital imaging techniques for observation of temporal and spatial interactions of multiple immune cell types with tumors using traditional fluorophores are limited by difficulties in concomitantly imaging multiple colors. We propose to solve this problem by developing and optimizing infusion protocols in mice using novel nanocrystal-antibody complexes, which should allow us to distinguish simultaneously five or more specific probes by three-dimensional intravital multiphoton laser-scanning microscopy. Information from this grant may elevate the clinical utility of immunotherapy approaches for cancer.